Method for producing cell lines and organs by means of differentiable cells

ABSTRACT

A method for producing cell lines or individual organs, differentiable donor cells being supplied to a morula or blastocyst which is cultivated under conditions that ensure a further development of the morula or blastocyst in stages in which newly formed cell lines having a higher degree of differentiation occur, as well as isolation of the cell lines or further differentiation of the cell lines into organs. The method according to the present invention is distinguished in that the cells of the morula or the internal cell mass of the blastocyst have a restricted survivability in comparison to the particular wild type or their survivability is reduced through suitable cultivation conditions, and the donor cells supplied to the morula or blastocyst have varying degrees of differentiation. A significantly shortened cultivation period of the donor cells may thus be achieved in comparison to typical methods, through which the provision of natural stem cells is favored, and perhaps made possible for the first time. The blastocysts may also be used by being transferred into a surrogate mother animal.

The present invention relates to a method for producing cell lines andorgans with the aid of differentiable cells according to the preamble ofclaim 1.

Pluripotent cells, as occur in early stages of embryonic development, aswell as cells of the germ line, are a special type of differentiablecells. In the following, pluripotent cells are understood as cells whichmay differentiate into every cell type. A property comparable topluripotence, but probably based on plasticity, may, however, also beinduced in cells of later development stages through technical measures,such as those according to the method according to the presentinvention. These cells are not included in the following by the term“pluripotent cells”. Pluripotent cells have the capability of producingall cell types of the embryo, the fetus, and the adult organism, as wellas regenerating themselves nearly infinitely. A renewable source ofcells which may be differentiated into manifold different types oftissue certainly offers manifold application possibilities in basicresearch and in transplant therapy. An important step for implementingthis goal is represented by the discovery that human, embryonic stemcells may be cultivated [3]. In the following, “embryonic stem cells”are understood as pluripotent cells which are removed from a morula orblastocyst and preferably kept viable in culture dishes. Obtaining andcultivating them is well known to those skilled in the art and wasdisclosed, for example, in U.S. Pat. No. 6,011,197 and WO 97/37009.These embryonic stem cells (ES cells) are primarily obtained in thiscase from the internal cell mass of blastocysts, i.e., those cells ofthe blastocysts from which all cells of the later endoderm, ectoderm,and mesoderm finally arise. It is noted at this point that in thefollowing a “pre-embryo” is understood as a developing cell mass up today 6 after fertilization of the egg cell, and after day 6, andtherefore possibly after implantation in the birth mother, reference ismade to an “embryo”. In particular, the term “pre-embryo” here comprisesthe preimplantation stages from the zygote via the morula up to theblastocyst until day 6 after fertilization of the egg cell. The embryoin the single-cell stage (pronucleus stage) is identified here as azygote. Preimplantation stages may be initiated through fertilization ofan egg cell by a sperm, through parthenogenetic activation of an eggcell, or through addition of one or more blastomers into an inductiveenvironment such as a zona pellucida, as was described by Alikani andWilladsen [11]. The term morula refers here to all stages of celldivisions following the zygote, including the early cell division stageson days 2 and 3, in which a blastocoel has not yet formed. Afterformation of a blastocoel, reference is made in the following to ablastocyst, this term also able to refer to early embryonic stages. Theblastocysts are formed by the zona pellucida (external, non-cellularmass) and by the trophoblasts and contain the internal cell mass alreadycited. After the hatching from the zona pellucida around day 6, thisstructure is also referred to as the blastocyst and is an embryoaccording to the above terminology. Methods for isolating an internalcell mass from a blastocyst are known to those skilled in the art [8,9]. “Embryonic stem cells” or “ES cells” would therefore actually be“free embryonic stem cells” according to this terminology, if they wereobtained from the morula or the blastocyst up to day 6, but the term“embryonic stem cells” or “ES cells” is generally maintained forpluripotent stem cells which were obtained from the morula orblastocyst, even if the removal is to be performed before day 6.

The potential of pluripotent cells such as ES cells for research andclinical use is extensive. Their future significance for in vitrostudies of human embryogenesis, for investigations of abnormaldevelopment (for example, through the production of cell lines havingintentional gene alterations), for the investigation of the effect ofindividual genes, for the development and testing of novel medications,or as a renewable source for cell and tissue transplants or for genetictherapies may currently hardly be estimated.

A further impetus for research of this type is provided by theobservation that ES cells of a first species may be introduced into theblastocyst of a second species, even a different one, which, aftertransfer into a female of the second species, leads to the birth of anoffspring that combines genetic features of both species and thusrepresents a chimera, which is also understood in the following as adeveloping cell mass that contains a subgroup of cells which have DNAhaving significantly different nucleotide base sequences in the cellnuclei than the other cells of the cell mass.

In order to elevate the genetic contribution of the donor ES cells tothe finally resulting organism, it was suggested in US 2002062493, forexample, that non-human mammals be produced by injecting ES cells of therelevant animal into tetraploid blastocysts of the same species. Theblastocysts are cultivated until development of an embryo andtransferred to a female to be carried until delivery. In a furtherembodiment, the ES cells are provided with mutations and then injectedinto tetraploid blastocysts, through which offspring having intentionalmutations may be generated. Methods of this type have been ascribed witha large potential for the research of the effect of individual genesand/or their mutations on the phenotypic development of offspring.

To produce tetraploid blastocysts, typically the blastomers of diploidpre-embryos in the two-cell stage are fused by applying brief electricalpulses. The embryos thus arising may be cultivated and result in thedevelopment of morulae and blastocysts, the tetraploidy manifesting inthe cells of the internal cell mass, for example, in the latter.

Morulae may be used for aggregation with ES cells and blastocysts forinjection of ES cells, for example. However, tetraploid blastocysts aredistinguished by a restricted development capability. While thedifferentiation of tetraploid cells hardly exceeds the development ofearly endoderm and trophoectoderm, injected diploid ES cells may resultin the development of a mature embryo, for example. Therefore, thesemethods were suggested, as described in US2002062493, for example, inorder to produce chimeras effectively, since due to the reduced lifespanof the tetraploid cells of the internal cell mass, the unfolding of thephenotype of the host is suppressed naturally in favor of the ES cellsof the donor organism. In particular, it was suggested that thephenotypic contribution of specific genes be determined rapidly with theaid of genetically modified ES cells [1].

In this context, it is to be noted that all attempts to induce a normalfetal development by completely removing the internal cell mass andreplacing it by ES cells have been shown to be failures, although EScells have been ascribed with the capability of producing the entirefetus. Therefore, it is assumed that the internal cell mass of the hostapparently exerts a decisive function of inducing the ES cells toreenter into an embryonic differentiation program, even if the cells ofthe internal cell mass are tetraploid cells having restricted viability.

Obtaining, using, and genetically altering ES cells encounters ethicalconsiderations, particularly with human ES cells, so that alternativesare to be sought, both in regard to the use of human blastocysts andalso human ES cells in research and clinical therapy.

The use of embryonic stem cells also encounters technical difficulties,however. These cells may thus currently only be obtained frompre-embryos or very early embryos and are not immunologically compatiblewith most patients, although multiple ES cell lines have currently beenisolated. A possible explanation may be that human ES cells expressMHC-I [10]. Therefore, it will either be necessary to isolate multiplefurther ES cell lines or tailor ES cell lines to each patient with theaid of “therapeutic cloning”. Furthermore, ES cells tend to formteratomas after being transplanted. ES cells must therefore bedifferentiated reliably into appropriate tissue types during theircultivation, before being transplanted. In addition, the question arisesof whether specialized cells, which were derived from ES cells, alsohave the desired functional properties in corresponding tissue afterbeing transplanted. Thus, for example, it has been shown that mouse EScells which produced insulin in vitro could not cause any reduction ofthe blood sugar level in vivo.

Therefore, alternatives to the use of ES cells have entered the focus ofresearch interest. Thus, the question arises of whether adult stemcells, possibly even from umbilical cord blood, may also be used as areplacement for ES cells, since it appears obtaining them from samplesof adult organisms, for example, seems less ethically questionable, evenin humans, than obtaining ES cells from pre-embryos or early embryos.This initially appeared impossible because of the restricteddifferentiability of adult stem cells.

As the most recent investigations have shown, however, MAPCs(“multipotent adult progenitor cells”) may be obtained upon purifyingmesenchymal stem cells of mice, which differentiate not only intomesenchymal cells, but rather also into cells of the endoderm, mesoderm,or ectoderm [2]. If MAPCs are injected into early mouse blastocysts, forexample, it may be determined that they contribute to the formation ofmultiple, possibly even all somatic cell types. These investigations areinteresting in that it had been assumed until now that tissue-specificstem cells, which surely have less capability for self-regenerationavailable than ES, generally differentiate only into cells of therelevant tissue. It has been observed that hematopoietic stem cells mayalso differentiate into cells of other tissue types under certaincircumstances or that neuronal stem cells may contribute to multipletissue types after injection into a blastocyst, but it was typicallythought to be practically impossible for a single tissue-specific stemcell to be able to differentiate into functional cells of multipletissue types. As the investigations have also been able to show, MAPCsalso contribute in vivo to the formation of multiple somatic tissuetypes when they are administered to a mouse. In fact, it has been shownthat MAPCs are similar to ES cells in their properties and, in addition,represent a very synchronous cell type in regard to theirdifferentiability, as has been shown on the basis of investigations ofgene expression. They require approximately comparable cultureconditions to ES cells, express at least some of the genetic markerswhich are also observed in ES cells in vitro (Oct-4, Rex-1, SSEA-1), buthave pronounced proliferation and differentiation properties, possiblycontribute to the formation of all organs when they are injected intoblastocysts, and differentiate into tissue-specific cells when they aresubjected to the corresponding influence of the relevant organs.

The nature of these MAPCs is currently still unexplained, and it evenappears questionable whether the MAPCs observed in vitro, which are theresult of comparatively long cultivation periods of multiple months,also exist in this form in vivo. Thus, it has been speculated that MAPCsactually do not exist in vivo, but rather that cells having alteredproperties, in some circumstances also similar to those of cancer cells,were cultivated. According to a further explanation model, the longcultivation period may encourage the reduction of the original cellpopulation to included stem cells, as was observed in the hematopoieticcells, which are very similar in their properties.

Finally, it was disclosed in EP 1176189 that cells may be obtained fromsamples of adult somatic cells, from muscle tissue, brain tissue, theblood, the bone marrow, the liver, or the mammary glands, for example,which display behavior similar to the pluripotent stem cells, andparticularly display expression of Oct-4, as is also the case inpluripotent stem cells in early stages of embryonic development. Thesecells were also referred to as “de-differentiated” stem cells, in orderto thus express the suspicion that cells may apparently regain greaterdifferentiability. However, more precise statements about theirusability for the production of differentiated or novel, differentiablecells were not provided.

It is the object of the present invention to provide a method forproducing differentiated or novel, differentiable cell lines or evenentire organs, without using differentiable cells that were obtainedfrom pre-embryos or embryos, such as ES cells. It is a further object ofthe present invention to achieve this while avoiding long cultivationperiods of the differentiable cells used. Notwithstanding the risksalready noted of long cultivation periods in connection with theabove-mentioned MAPCs, hazards are also outlined in this regard on thebasis of “genetic imprinting” [4, 5].

The object of the present invention is achieved by the characterizingfeatures of claim 1.

In claim 1, to produce differentiated or novel, differentiable celllines or even organs, embryonic stem cells or stem cell lines which areto have a uniform degree of differentiation in regard to theirpluripotence, are not used, as is described in U.S. Pat. No. 6,200,806or in [12], but rather cells having a primary varying degree ofdifferentiation, which particularly characterizes a sample of a donororganism containing adult, somatic stem cells. In this case, “varyingdegree of differentiation” of donor cells is understood to mean thatthey may comprise multipotent/pluripotent or even differentiated cells,a relatively large number of differentiated cells or cells which canhardly be differentiated further typically being found in a sample ofadult cells of a donor organism and only a small number of cells stillhaving multipotent/pluripotent character. The donor cells are certainlycell populations which have experienced preparation with the aid ofsuitable methods to increase the concentration of included stem cells,for example, in the course of the production of a highly-purifiedfraction from umbilical cord blood, but obtaining synchronous cellpopulations, i.e., cells having a uniform degree of differentiation, asis the case when obtaining embryonic stem cell lines, and the longcultivation period connected therewith, is dispensed with.

Even if it was conceivable to synchronize the sample in regard to thedifferentiability of the included cells with the aid of sufficientlylong cultivation periods, this is not necessary if it is introducedaccording to the present invention into morulae or blastocysts, whosecells, in blastocysts those of the internal cell mass, have asurvivability restricted in comparison to the wild type morula and/orwild type blastocyst, or if the survivability of these cells is reducedthrough suitable cultivation conditions.

In the following, a wild type morula and/or wild type blastocyst isunderstood as a morula or blastocyst which has not yet experienced anymanipulation in this connection. As was already noted, the internal cellmass of the host blastocyst apparently exerts a decisive function inregard to inducing the ES cells introduced into the blastocyst toreenter an embryonic differentiation program, even if the cells of theinternal cell mass are cells having restricted survivability, because oftetraploidy, for example. As has now surprisingly been determined, it ispossible through of the “reprogramming” cell matrix provided by thecells of the internal cell mass, but also the cells of the morula, tocause a “de-differentiation” in regard to greater differentiability evenin non-embryonic stem cells. It is also conceivable that it is moreappropriate to speak of a “trans-differentiation” of the supplied donorcells, their plasticity being caused to unfold by being embedded in anappropriately stimulating cellular environment. The mode of operation ofthe “stimulation” is currently not completely explained, but bothintercellular factors, such as autocrine and paracrine factors, and alsothe “polarity” of the embryo appear to play a role, in addition to theextracellular matrix. The present invention is therefore based on theidea that possible deficits in regard to the differentiability of stemcells which were not obtained from pre-embryos or early embryos may becompensated for through their contact with an appropriatelyreprogramming cell matrix, consideration having to be taken that, inregard to an optimized yield of newly-formed cell lines, the cells ofthe internal cell mass of the blastocyst and/or the cells of the morulamust have a restricted survivability in comparison to the wild typeblastocyst and/or wild type morula, or their survivability must bereduced through suitable cultivation conditions. The cells of theinternal cell mass and/or the cells of the morula therefore support thedesired reprogramming of the donor stem cells supplied, although theirproportion in the cells of the developing organism is continuouslyreduced because of their restricted survivability.

“Restricted survivability” of the cells of the morula and/or internalcell mass of the blastocyst may be produced in different ways. Eitherthe restricted survivability is already provided “intrinsically”, as intetraploid embryos, or restricted survivability of certain cells may beinduced “extrinsically” with the aid of suitable cultivation conditions.Both possibilities will be discussed in the following.

Claim 2 provides a preferred embodiment, according to which the donorcells contain naturally occurring stem cells. “Naturally occurring” stemcells are understood here as adult, somatic stem cells, also fromumbilical cord blood, as may be found in vivo. In regard to theabove-mentioned MAPCs, it was determined that MAPCs isolated after longcultivation periods of multiple months may not actually exist in vivo,but rather cells having altered properties were cultivated because ofthe long cultivation period. It is also not entirely to be excluded inthe method according to the present invention that, in spite of thesignificantly shorter preparation and cultivation periods, the donorcells represent a cell population altered in comparison to the naturalstem cells occurring in vivo. In the preferred embodiment of the methodaccording to the present invention, the donor cells contain naturallyoccurring stem cells, however, which is also favored by a rapidpreparation and cultivation of the donor cells, in addition to the useaccording to the present invention of donor cells having varying degreesof differentiation.

Claim 3 provides a special embodiment of the method according to thepresent invention, according to which the cells of the morula or theinternal cell mass of the blastocyst are prepared as a reaction mediumin a culture dish. However, it would also be conceivable to use thecells of the morula or the internal cell mass of the blastocyst toprepare a reaction medium as described in [37], since it is currentlyassumed that dissolved matrix components such as laminin, collagen IV,cytokine, or even proteins existing on the cells, such as glycoproteins,may also act as a “reprogramming” cell matrix. A standard medium ispreferably used for this purpose, which preferably dispenses with theuse of FCS (“fetal calf serum”) or another serum derived from animalproteins.

According to claim 4, the donor cells are obtained from umbilical cordblood, particularly by producing a highly purified fraction whichcontains approximately 5% stem cells.

According to claim 5, the donor cells are obtained from the placenta.The placenta contains multiple cells which are of interest for themethod according to the present invention, such as mesenchymal cells andendothelial cells, which are assumed to be an especially bountifulsource for stem cells. According to claim 6, the donor cells areobtained from the bone marrow. According to claim 7, the donor cells areobtained from the fatty tissue, which is distinguished as an especiallyinteresting source, since stem cells from the fatty tissue arerelatively easy to obtain. Containing the donor cells comprisespreparing the sample, taken from an adult organism, the preparationhaving the goal of elevating the concentration of stem cells containedin the sample. Methods of this type are related art and are well knownto those skilled in the art.

According to claim 8, the cells of the receiver morula and/or theinternal cell mass of the receiver blastocyst are tetraploid cells.Methods for implementing such a tetraploidy are known according to therelated art [6, 7]. As has already been noted, tetraploid cells have arestricted survivability. Because of the restricted survivabilityintrinsically provided in the tetraploid cells of the morula and/or theinternal cell mass of the blastocyst, their number is gradually reducedand an increasing popularization of the developing blastocyst withsuccessor cells of the donor cells supplied is thus ensured, thetetraploid cells setting the required intercellular signals forreprogramming the donor cells supplied in regard to greaterdifferentiability over the duration of their existence.

Claim 9 provides an alternative method for equipping the cells of themorula or the internal cell mass of the blastocyst with a restrictedsurvivability, in that a vector is incorporated into their genome whichcauses a lethal sensitivity to appropriately selected cultivationconditions. If the cultivation conditions are selected accordingly afterthe donor cells are supplied to the morula and/or the blastocyst, thisresults in intentional dying of the cells of the morula and/or theinternal cell mass, without impairing the survivability of the donorcells and the trophoblasts. Thus, for example, vectors may be selectedwhich cause a higher sensitivity to temperature increase or specificadditives for culture media.

As an alternative to this, according to claim 10, the genome of thedonor cells is provided with a vector such as a neomycin-resistance geneor puromycin-resistance gene, which causes a resistance to media havingadditives such as G 418 or puromycin. However, it must be ensured forthis purpose that the incorporation of the vector into the donor cellsdoes not cause cultivation times that impair the object according to thepresent invention of the shortest possible cultivation times of thedonor cells before they are supplied to the blastocyst. Vectors may alsobe supplied which make the cells resistant to specific temperatureinfluences, such as temperature increases [13-17].

As a further alternative, according to claim 11 the survivability of thecells of the morula and/or the internal cell mass of the blastocyst isreduced by adding suitable antibodies to the culture media. By addingspecific antibodies (AB), which are charged with cell-damagingsubstances and only adhere to cells that have a receptor for thespecific AB, only these cells are thus damaged. Techniques of this typeare described, for example, in [18-22].

Implementing claims 9 through 11 allows an advantageous embodiment ofthe method according to the present invention according to claim 12 tobe implemented, according to which the reduction of the survivability ofthe cells of the morula and/or the internal cell mass of the blastocystis performed in a way which is tailored to the varying degrees ofdifferentiation of the donor cells and is chronologically well-ordered.A varying composition of the donor cells introduced into the host morulaand/or host blastocyst may specifically require the cells of the morulaand/or the internal cell mass of the blastocyst to die in achronologically tailored way, in order to achieve an optimized signalsetting of the reprogrammed cell matrix. The intentional reduction ofthe survivability of the cells in the morula and/or the internal cellmass must ensure, however, that the trophoblasts are not impaired intheir survivability, since they are important for the further survivalof the embryo, particularly if the blastocysts are transferred into asurrogate mother animal.

The cell sample obtained from a cell sample of the donor organism orfrom umbilical cord blood represents, as noted, even after purificationof the sample in regard to the differentiability of the included cells,an asynchronous cell population, which not only contains multipotent, atbest even pluripotent stem cells, but rather also cells having lesserdifferentiability, such as tissue-specific cells, which may complete atrans-differentiation into tissue-specific cells of other tissues underspecific circumstances, however, or even differentiated cells possiblywithout any differentiability. Therefore, it may be advantageous tobring the donor cells into contact with other blastocysts or isolatedinternal cell masses of other blastocysts in culture dishes according toclaim 13 before the donor cells are supplied to the morula and/orblastocyst. Methods for isolating and cultivating internal cell massesare known to those skilled in the art, a medium of undifferentiatedcells finally being prepared from isolated internal cell masses, ontowhich the donor cells may be applied or washed with a high probabilityof contact with the cells of the prepared internal cell mass.Specifically, it has been shown that through the contact withblastocysts or isolated internal cell masses of blastocysts, a selectionof cells may be achieved which is suitable for the method according tothe present invention in regard to higher differentiability. Donor cellshaving a relatively high affinity to the medium prepared from internalcell masses or the blastocysts may be isolated and are available forfurther therapeutic, diagnostic, or scientific applications, but mayalso be injected into morulae or blastocysts for furtherdifferentiation. In the latter case, the probability of induction of ahigher differentiability of the injected donor cells by the cell matrixof the morula and/or the internal cell mass of the host blastocysts iselevated, the duration of this additional method step only being a fewminutes, or even a few seconds if the donor cells are washed onto themedium prepared from internal cell masses, so that even if this step isimplemented, the cultivation periods may be kept comparatively short.

As an alternative to this, the features of claim 14 may also beimplemented, according to which cells suitable for the method accordingto the present invention are preselected via appropriate markers.

Claims 15 and 16 provide special embodiments of the method according tothe present invention. Particularly if human donor cells are used andare injected into pig blastocysts, for example, the possibility arisesvia the method according to the present invention of producing celllines whose genetic properties are comparable, and in the best caseidentical, to those of the original, human donor cells, although the pigblastocyst developing after application of the method according to thepresent invention is in no way genetically identical to the donor cells.In a later development stage of the pig blastocyst, novel,differentiable cells, differentiated cell lines, or even entire organshaving genetic identity to the human donor cells and in the optimum casealso immunological compatibility with the donor organism may beisolated, without this requiring the use of human embryonic stem cells.

Claim 17 provides an advantageous type of the supply of the donor cellsinto the host blastocyst, in that the supply is performed throughinjection.

Claim 18 provides an advantageous type of the supply of the donor cellsinto the host morula, in that the supply is performed throughaggregation.

Claim 19 relates to a special embodiment of the method according to thepresent invention, according to which the donor cells are human cells.However, it is thoroughly possible for the morula or blastocysts towhich the donor cells are supplied to nonetheless be of non-humanorigin. Since the cell lines or even organ structures harvested from themethod according to the present invention are genetically identical tothe donor cells, they are suitable for use as a preparation fortherapeutical intervention according to claims 20 and 21, for example,for illnesses as are cited in claim 22.

Claim 23 relates to a special embodiment of the method according to thepresent invention, according to which the donor cells are non-humancells. Since the cell lines harvested from the method according to thepresent invention are again genetically identical to the donor cells,they are suitable for use as a preparation for therapeutic anddiagnostic intervention in the veterinary field according to claim 24,and for producing genetically identical cells and organ structures fortherapeutic, diagnostic, or scientific application according to claim25, for illnesses as are cited in claim 26, for example.

A possible embodiment of the method according to the present inventionwill now be described in greater detail on the basis of the attachedFIGS. 1 through 3.

For this purpose, FIG. 1 is to schematically illustrate how, accordingto an embodiment of the method according to the present invention, atetraploid blastocyst 1 is first produced. The blastomers 2, surroundedby the zona pellucida 3, of a two-cell pre-embryo may be convertedthrough electrofusion, for example, into a one-cell pre-embryo having atetraploid chromosome number. Techniques for producing tetraploidpre-embryos are known from the related art and described, for example,in [6, 7, 23-27].

Furthermore, the pre-embryo completes cell divisions of the blastomersand develops further into a blastocyst 1. The internal cell mass 4 andthe trophoblasts 5 are indicated schematically in FIG. 1.

In addition, a sample originating from umbilical cord blood, forexample, or even a sample taken from an adult organism, from fattytissue, for example, is subjected to a preparation which is intended toelevate the concentration of the included stem cells. Techniques forpreparing a sample for the purpose of producing a purified cell fractionare also known according to the related art and described, for example,in [28, 32, 34]. The result of the preparation is donor cells 6 (FIG.2), which have varying degrees of differentiation and may comprisemultipotent/pluripotent or even differentiated cells, a relatively largenumber of differentiated or hardly still differentiable cells beingfound in a sample of adult cells of the donor organism and only a smallnumber of cells still having multipotent/pluripotent character. Thedifferentiation potential is known for some types of adult stem cellsaccording to the related art [e.g., 29, 30, 31, 34].

In the framework of the method of according to the present invention,the donor cells 6 are not synchronized in regard to thedifferentiability of the included cells with the aid of sufficientlylong cultivation periods, but rather they are introduced into morulae 7or blastocysts 1, whose cells 2, in blastocysts those of the internalcell mass 4, now have restricted survivability, because of thetetraploidy produced, in comparison to the wild type morula and/or wildtype blastocyst (FIG. 3). As was already noted, the cells 2 of the hostmorula 7 and/or the internal cell mass 4 of the host blastocyst 1apparently exert a decisive function in inducing introduced stem cellsto reenter an embryonic differentiation program. As has now surprisinglybeen determined, it is possible because of the “reprogramming” cellmatrix provided by the cells of the internal cell mass 4, but also bythe cells 2 of the morula 7, to cause a “de-differentiation” in regardto greater differentiability, even in non-embryonic stem cells. As wasalready noted, it may be more relevant to speak of a“trans-differentiation” of the supplied donor stem cells 6, theirplasticity being caused to unfold by embedding them in an appropriatelystimulating cellular environment. The present invention is thereforebased on the idea that possible deficits in regard to thedifferentiability of stem cells which were not obtained from pre-embryosor early embryos may be compensated for through their contact with anappropriately reprogramming cell matrix, care having to be taken that,in consideration of an optimized yield of newly-formed cell lines, thecells of the internal cell mass 4 of the blastocyst 1 and/or the cells 2of the morula 7 have a restricted survivability in comparison to thewild type blastocyst and/or wild type morula. The cells of the internalcell mass 4 and/or the cells 2 of the morula 7 thus do support thedesired reprogramming of the supply donor stem cells 6, although theirproportion in the cells of the developing organism continuouslydecreases because of their restricted survivability. As is alsoindicated in FIG. 3, the cells 2 of the morula 7 may be prepared in aculture dish 8 in this case. As an alternative to this, the cells of theinternal cell mass 4 of the blastocyst 1 may also be prepared in aculture dish 10. Furthermore, it is also conceivable to perform thecocultivation in a culture dish 9 with the aid of appropriately preparedblastocysts 1. Techniques for the cocultivation of stem cells with othercell types are described, for example, in [35, 36].

Particularly if human donor cells 6 are used and are injected, forexample, into pig blastocysts 1, the possibility arises via the methodaccording to the present invention of producing cell lines whose geneticproperties are comparable, and in the best case identical, to those ofthe original, human donor cells 6, although the pig blastocyst 1developing after application of the method according to the presentinvention is in no way genetically identical to the donor cells 6. In alater development stage of the pig blastocyst 1, novel, differentiablecells or differentiated cell lines having genetic similarity and/oridentity to the human donor cells 6 and in the optimum case alsoimmunological compatibility to the donor organism may be isolated,without this requiring the use of human embryonic stem cells.Preparations for multiple human illnesses, as listed in the claims, maybe manufactured from these cell lines.

Upon transfer of the pig blastocyst 1 into a surrogate mother animal, itis also conceivable to allow the development of organs with geneticsimilarity and/or identity to the human donor cells 6 and in the optimumcase also immunological compatibility to the donor organism. Thus, withthe aid of the method according to the present invention, for example, aheart of a pig made of up to 100% human cells may result from a humandonor cells 6 which were obtained without the use of a pre-embryo orembryo, using a pig blastocyst, without causing suffering of theaffected animal in this case. In the optimum case, the heart would beavailable with complete immunological compatibility to the donororganism.

LITERATURE

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1. A method for producing cell lines or individual organs,differentiable donor cells (6) being supplied to a nonhuman morula (7)or nonhuman blastocyst (1), which are cultivated under conditions thatensure a further development of the morula (7) or blastocyst (1) instages in which newly formed cell lines having a higher degree ofdifferentiation occur, and comprising the isolation of the cell lines orfurther differentiation of the cell lines into organs through transferof the blastocyst (1) into a surrogate mother animal, wherein the cells(2) of the morula (7) or the internal cell mass (4) of the blastocyst(1) have a restricted survivability in comparison to the particular wildtype or their survivability is reduced through suitable cultivationconditions, and the donor cells (6) supplied to the morula (7) orblastocyst (1) have varying degrees of differentiation and are ofnon-embryonic origin.
 2. The method according to claim 1, wherein thedonor cells (6) contain naturally occurring stem cells.
 3. The methodaccording to claim 1, wherein the cells (2) of the morula (7) or theinternal cell mass (4) of the blastocyst (1) are prepared in a culturedish (8, 9, 10) or are used to prepare a soluble matrix fraction.
 4. Themethod according to claim 1, wherein the donor cells (6) are obtainedfrom umbilical cord blood.
 5. The method according to claim 1, whereinthe donor cells (6) are obtained from placenta.
 6. The method accordingto claim 1, wherein the donor cells (6) are obtained from bone marrow.7. The method according to claim 1, wherein the donor cells (6) areobtained from fatty tissue.
 8. The method according to claim 1, whereinthe cells (2) of the morula (7) or the internal cell mass (4) of theblastocyst (1) are tetraploid cells.
 9. The method according to claim17, wherein the cells (2) of the morula (7) or the internal cell mass(4) of the blastocyst (1) has cells whose genome contains vectors thatcause a lethal sensitivity to appropriate cultivation conditions incomparison to the particular wild type.
 10. The method according toclaim 1, wherein the genome of the donor cells (6) contains a vectorwhich causes a resistance to additives of culture media.
 11. The methodaccording to claim 1, wherein the survivability of the cells (2) of themorula (7) or the internal cell mass (4) of the blastocyst (1) isreduced by adding suitable antibodies.
 12. The method according to claim9, wherein the survivability of the cells (2) of the morula (7) or thecells of the internal cell mass (4) of the blastocyst (1) is reduced ina way that is tailored to the varying degrees of differentiation of thedonor cells (6) and is chronologically well-ordered.
 13. The methodaccording to claim 1, wherein before the donor cells (6) are suppliedinto the morula (7) or the blastocyst (1), the donor cells (6) arebrought into contact in culture dishes with other blastocysts orinternal cell masses isolated from other blastocysts, and those donorcells having a relatively high contact affinity are isolated andsupplied to the morula (7) and/or blastocyst (1) first cited.
 14. Themethod according to claim 1, wherein before the donor cells (6) aresupplied into the morula (7) or the blastocyst (1), the donor cells (6)are equipped with a genetic marker that ensures cells having a lowerdegree of differentiation are isolated and supplied into the morula (7)or blastocyst (1).
 15. The method according to claim 1, wherein themorula (7) or blastocyst (1) is a mouse morula or mouse blastocyst. 16.The method according to claim 1, wherein the morula (7) or blastocyst(1) is a pig morula or pig blastocyst.
 17. The method according to claim1, wherein when the donor cells (6) are supplied to a blastocyst (1),the supply is performed through injection.
 18. The method according toclaim 1, wherein when the donor cells (6) are supplied to a morula (7),the supply is performed through aggregation.
 19. The method according toclaim 1, wherein the donor cells (6) are human donor cells. 20.(canceled)
 21. (canceled)
 22. (canceled)
 23. The method according toclaim 1, wherein the donor cells (6) are donor cells of non-humanmammals.
 24. (canceled)
 25. (canceled)
 26. (canceled)